text: Aza-IP 5-azacytidine-mediated RNA immunoprecipitation has been optimized on and used for detecting targets of methyltransferases, particularly NSUN2 and DNMT2 — the two main enzymes responsible for laying down the m5C mark.
  First, the cell is made to overexpress an epitope-tagged m5C-RNA methytransferase derivative so that the antibody used later on for immunoprecipitation could recognize the enzyme. Second, 5-aza-C is introduced to the cells so that it could be incorporated into nascent RNA in place of cytosine. Normally, the methyltransferases are released (i.e. covalent bond between cytosine and methyltransferase is broken) following methylation of the residue. For 5-aza-C, due to a nitrogen substitution in the C5 position of cytosine, the RNA methytransferase enzyme remains covalently bound to the target RNA molecule at the C6 position.
  Third, the cell is lysed and the m5C-RNA methyltransferase of interest is immunoprecipitated along with the RNA molecules that are covalently linked to the protein. The IP step enabled >200-fold enrichment of RNA targets, which were mainly tRNAs. The enriched molecules were then fragmented and purified. cDNA library is then constructed and sequencing is performed.
  An important additional feature is that RNA methyltransferase covalent linkage to the C5 of m-aza-C induces rearrangement and ring opening. This ring opening results in preferential pairing with cytosine and is therefore read as guanosine during sequencing. This C to G transversion allows for base resolution detection of m5C sites. 
  One caveat is that m5C sites not replaced by 5-azacytosine will be missed.